Poly (I:C) increases the expression of galectin 1, 3, 9 and HGF genes in exosomes isolated from human Wharton's jelly mesenchymal stem cells

Background Mesenchymal stem cells (MSCs) are commonly employed as a powerful tool for the treatment of immune-mediated problems owing to their capacity to regulate the immune system and differentiate into different tissues. Researchers use mesenchymal stem cell products given the limitations associated with the application of MSCs. Exosomes are nanometer vesicles derived from MSCs that are used in cell-free therapy. Inflammatory environmental conditions, such as stimulation of Toll-like receptor 3 (TLR-3), has the ability to adjust the immune-regulating properties and anti-inflammatory function of mesenchymal stem cells and their exosomes. Galectins and hepatocyte growth factor (HGF) are known as immunomodulatory factors in mesenchymal stem cells. This study was designed to examine the expression of galectin-1, galectin-3, galectin-9, and HGF genes in exosomes isolated from human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) after stimulation with Poly (I:C) (Polyinosinic:polycytidylic acid sodium salt). Methods To begin, the explant technique was used to extract mesenchymal stem cells from human umbilical cord Wharton's jelly. Then, the stem cells were stimulated using Poly (I:C) at three time intervals of 12, 24 and 48 h. Exosomes secreted from the supernatant of cells were extracted and exosome confirmation tests, including Scanning electron microscopy (SEM), Dynamic light scattering (DLS) and Flow cytometry were performed. Finally, the expression of galectin-1, galectin-3, galectin-9, and HGF genes in exosomes was evaluated by Real-Time PCR at three time intervals of 12, 24 and 48 h after stimulation. Results The findings of our study indicated that following stimulation with Poly (I:C), the expression of galectin-9 and HGF (P < 0.05) genes was markedly higher than in the control group after 12 h. After 24 h, the expression of galectin-9 (P < 0.01), galectin-3 and HGF (P < 0.05) increased; the expression of galectin-1, galectin-3, (P < 0.05), galectin-9 and HGF genes (p < 0.01) significantly increased compared to the control group after 48 h. Conclusion TLR3 stimulation can increase the expression of galectins and HGF genes in exosomes derived from hWJ-MSCs and may be improve the immunosuppressive abilities of exosomes.

Mesenchymal stem cells also express anti-inflammatory genes like HGF, galectin-1, 3, 4, 8, 9, which inhibit immune responses [9,10].There are fifteen members of the galectin family in humans, and eleven of them are expressed in different human tissues [11].The carbohydrate recognition domains (CRDs) are shared by all galectins and bind to β-galactosides more preferentially.Both the innate and adaptive immune systems rely on galectins to maintain cellular homeostasis [12].The suppressive effect of MSCs on allogeneic T-cells was reversed when galectin-1 and galectin-3 gene expression was inhibited using small interfering RNAs (siRNA), suggesting that galectins, especially galectin-1 and galectin-3, play a role in the immunomodulation of MSCs [13][14][15].Galectin-1 has an inherent expression in mesenchymal stem cells and inhibits the immune system; however, Galectin-9 has an inducible expression and increases sharply under the influence of inflammatory conditions [16].MSCs suppress and inhibit antigen presentation in antigen presenting cells such as dendritic cells by secreting HGF.
Biological properties of MSCs can be modulated by the inflammatory microenvironment.Interestingly, mesenchymal stem cells exhibit anti-inflammatory function under inflammatory conditions and increase the expression of anti-inflammatory genes [17,18].Therefore, several strategies have been sought to intensify the immunomodulatory function of MSCs and obtain more consistent results.Priming, or preconditioning, is an important method for improving the immunological and antimicrobial capabilities of MSCs ex vivo, which in turn improves their ability to modulate immune responses [19].Among these approaches, the priming and licensing of MSCs with Toll-like receptors (TLRs) agonists have been extensively studied [20,21].
Pattern recognition receptors such as Toll-like receptors are able to identify several molecular patterns that are linked with pathogens.MSCs express functional Toll-like receptors that can affect the phenotype, multilineage potential and immunomodulatory capacity of MSCs.Studies have shown that TLR3 acts as the first mediator of anti-stress responses in human mesenchymal stem cells (HMSCs) [22].Stimulation of these cells with Poly (I:C), a TLR3 ligand, enhances their migration to the stress site [23].It has been shown that TLR3-primed MSCs express immunosuppressive factors such as IDO, prostaglandin E2 (PGE2), nitric Oxide (NO), transforming growth factor beta (TGF-β), heme Oxygenase (HO), and hepatocyte growth factor (HGF) [24,25].
Due to the problems related with MSCs such as tumor formation and spontaneous differentiation, researchers proposed the application of MSCs secreted products in the culture medium.Extracellular vesicles (EVs) are among the components secreted by MSCs.Exosomes (Exo) are extracellular vesicles, namely small membranous vesicles (30-150 nm) that participate in cell-cell interactions [18].Exosomes are released by nearly all cell types.Among the many surface markers carried by these vesicles are the ubiquitously expressed CD9, CD63, and CD81.The protein, lipid, and RNA expression of exosomes from different cells and organisms has been extensively described in ExoCarta database [26].Based on recent research, it appears that MSC-EXO, which are exosomes derived from MSCs, exert an inhibitory effect on the immune response, including effects on T-cells, B cells, inflammatory cytokines, and tolerogenic signaling [27].This suggests that MSC-EXO could play a significant role in the immunomodulatory functions of MSCs and offer a safer and more repeatable treatment option compared to MSCs themselves.Exosomes are essential in controlling immunological responses because of their ability to both stimulate and suppress the immune system [27].
There is a lack of data in literature regarding the impact of MSCs on immunosuppressive properties of exosomes generated from them.With this in mind, in the present work, we evaluated the effect of Poly (I: C) on the expression of anti-inflammatory genes (galectin -1, galectin -3, galectin -9, and HGF) in MSCs-derived exosomes.

Isolation and expansion of hWJ-MSCs
Human umbilical cords (UC) were collected from healthy full-term infants delivered by cesarean section with the consent of mothers who were also negative for hepatitis B, C, HIV, and syphilis (N = 10).Segments with a size of 5-10 cm were sectioned and preserved at 4 • C within sterile phosphate-buffered saline (PBS), 300 U/mL penicillin (Sigma), 300 μg/mL streptomycin (Sigma) and 7.5 μg/mL amphotericin B (Sigma) in aseptic condition.This study was approved by Ethics Committee of Ahvaz Jundishapur University of Medical Sciences (Ethics NO: IR.AJUMS.MEDICINE.REC.1398.030).
The umbilical cords were chopped into smaller 3-4 cm pieces and washed gently several times with phosphate-buffered saline (PBS) containing 100 U/mL penicillin (Sigma) and 100 μg/mL streptomycin (Sigma) to remove the blood.Then, the vein and arteries of umbilical cord were exposed and removed from the inner matrix.Slices of tissue with 1-3 mm 3 size were cut from the exposed extracellular matrix of Wharton's jelly.Afterward, 9-12 pieces were transferred onto a T25 flasks culture dish (SPL, Korea) with Dulbecco's modified eagle medium containing nutrient mixture F-12 (DMEM-F12; Gibco) supplemented with 10 % (v/v) fetal bovine serum (FBS; Gibco), 100 U/mL penicillin (Sigma), 100 μg/mL streptomycin (Sigma) and 2.5 μg/mL amphotericin B (Sigma) at 37 • C, 95 % humidity and 5 % (v/v) CO 2 .The fragments were removed once 80 % confluence was achieved after 10 days.The adhering fibroblast-like cells were then trypsinized with 0.025 % trypsin with 0.02 % EDTA (Gibco) and transferred to a fresh flask for further extension.This study utilized the cells grown between passages 3 and 5.

Evaluation of WJ-MSCs differentiation
To investigate the differentiation capacity of isolated WJ-MSCs to osteocytes and adipocytes, 5 × 10 4 WJ-MSCs were cultured in sixwell plates.The cells were cultured in DMEM-F12 with 10 μM β-glycerolphosphate (Sigma), 10 μM dexamethasone (Sigma), and 50 μg/ mL ascorbic acid biphosphate (Sigma) to achieve osteogenic differentiation.For a total of 21 days, the medium was changed twice weekly.After that, Alizarin Red was used to stain the cells so that the mineralized matrix could be seen under the microscope.To trigger adipogenic differentiation, the cells were placed in DMEM-F12 that included 100 nM of dexamethasone (Sigma) and 50 μg/mL of indomethacin (Sigma).Over the course of three weeks, the medium was changed twice a week.Lastly, Oil Red was used to stain the cells such that cytoplasmic oil vesicles were visible under the microscope.

Poly (I:C) -priming of WJ-MSCs
WJ-MSCs were cultured in T75 flask culture dishes (SPL) containing DMEM-F12 supplemented with 10 % FBS until they reached 60 % confluency.Starvation method was used for removing FBS and obtaining pure exosomes from cells.Then, FBS was decreased from 10 % to 0 % over 4 days.For priming, polyinosinic-polycytidylic acid sodium salt [poly (I:C); P1530, Sigma-Aldrich, Canada)] was added into WJ-MSCs culture medium at a final concentration of 50 μg/mL with 0 % FBS for 12, 24 and 48 h.After that, the cell culture medium containing poly (I:C) was changed and the new medium replaced with 0 % FBS.The culture media were subsequently collected after at least 48 h for exosome isolation.All the above steps were carried out for the control group, except for adding poly (I: C).

Exosomes extraction and characterization
MSC-derived exosomes were isolated using an exosome isolation kit (Exospin, Cell Guidance Systems, LLC, MO, USA) according to manufacturer's instructions.Exosome morphology was observed using scanning electron microscopy (SEM) (KYKY-EM 3200), which was accomplished by fixing the exosomes on a slide with glutaraldehyde for 15 min.After washing with PBS, a cold graded ethanol series (15-30 min per change): 30 %, 50 %, 70 %, 80 %, 90 %, 100 % were used for dehydrating the samples.The samples were subsequently kept at room temperature to dry for 24 h.After gold-palladium sputtering, the samples were examined by SEM.Exosome size distribution, polydispersity index (PDI) and zeta potential were determined using dynamic light scattering (DLS) approach (Malvern Instruments, Malvern, UK) and immunophenotyping was conducted for CD9, CD63 and CD81 surface markers by flow cytometry.FITC-anti-human CD63 (Clone: H5c6), PE-anti-human CD81 (Clone: 5A6) and APC-anti-human CD9 (Clone: H19a) was used for immunophenotyping of exosomes.All the antibodies were purchased from BioLegend [28].

RNA isolation and gene expression
After thoroughly lysing the exosome samples on ice and vortexing, total RNA was extracted from them using RIPA Lysis Buffer (R0278, Sigma-Aldrich, Germany) in a 3:1 ratio.Lastly, after leaving the mixture at room temperature for 5 min, it was centrifuged at 15,000 rpm to obtain the supernatant.Following this step, RNA integrity and purity were assessed using electrophoresis and Nanodrop 2000 equipment from Thermo Fisher Scientific (Wilmington, DC, USA), respectively.Afterward, the reverse transcription was carried out using total RNA, and cDNA synthesis was done using revertAid First Strand cDNA Synthesis Kit (Thermo Fisher, Canada) with a final reaction volume of 30 μL following the instructions provided by the manufacturer.ABI STEP ONE PLUS, a real-time PCR machine, was used for the experiment.Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the internal control that was utilized to normalize gene expression.The 2 -ΔΔCT technique [34], which analyzes data, was utilized to investigate the expression of genes such galectin-1, galectin-3, galectin-9, and HGF.Lastly, we looked at fold changes or expression levels compared to the control.Table 1 shows the primer sequences [43].

Statistical analysis
Data were analyzed using Prism software version 8 (GraphPad, San Diego, CA); One-way ANOVA were used to assess group differences.Our data was nonparametric and to identify the difference between groups, we used Kruskal-Wallis test.We have used Dunn's multiple comparisons test as Post Hoc test for multiple comparisons and two-tailed T-test was used.Mean ± standard deviation was used to represent the data, and statistical significance was determined by P < 0.05.

WJ-MSC characterization
Fourteen days after removal of tissue fragments, the cells adhered to plastic dishes and showed a fibroblastic and single-spindle shaped morphology.After three weeks, WJ-MSCs colonies reached confluency (Fig. 1A).Cell surface marker analysis was performed by flow cytometry to characterize WJ-MSCs.The WJ-MSCs were positive for CD90, CD105 and CD73 but were negative for CD45, CD31 and CD34 (Fig. 2).

Differentiation of osteogenic and adipogenic function
According to Alizarin Red S and oil Red O staining, in vitro differentiation results confirmed numerous lipid vacuoles (intracellular lipid vesicles) after 21 days in WJ-MSCs that were visualized by oil Red O (Fig. 1C).Moreover, when the mentioned cells were induced to differentiate into osteoblasts, massive calcium depositions (extracellular calcium deposits) were observed after Alizarin Red staining (Fig. 1B), which indicated the mesodermal origin of isolated cells.

Exosomes characterization
Exosome size, PDI and zeta potential were measured using DLS.The average size of the isolated exosomes was 132 ± 2.45 nm, PDI of 0.043 ± 0.023 and zeta potential of − 28.1322 ± 5.583244.The results depicted in this image are based on the value of e, which has been reported to be approximately equal to 2.71 (Fig. 3A and B).Shapes were measured using SEM.SEM images revealed a uniform spherical shape for exosomes with no significant deformities (Fig. 4).Surface marker analysis was performed by flow cytometry to characterize exosomes.The exosomes were positive for CD9, CD63 and CD81 (Fig. 5).

Toxicity of Poly (I:C) on WJ-MSCs
To evaluate the cytotoxic activity of Poly (I:C) on WJ-MSCs, the cells were incubated with different doses (5-50 μg/mL) of Poly (I: C).After 12, 24 and 48 h, of incubation, cell viability was determined by the MTT assay.The viability was greater than 85 % at different concentration of Poly (I:C) even at a concentration of 50 μg/mL (Fig. 6).

Table 1
Sepuences of oligonucleotide primers used for amplification in real-time PCR.

Discussion
The unique immunosuppressive ability of MSCs has made these cells a powerful tool to suppress inflammation and the immune system, which can prevent tissue damage in chronic inflammation and autoimmune diseases [29].These features have turned these cells into unique candidates for cell therapy.However, the use of MSCs has a number of limitations, including decreasing ability of these cells to multiply during multiple passages, spontaneous differentiation into various cell lines during the passage process, and the need to use MSCs autologously in the treatment process [30].Recently, it has been found that MSCs autocrinely secrete a series of  Extracellular vesicles secreted from MSCs include different types based on size and content [31].Exosomes released from MSCs have all the genotypic and phenotypic characteristics of their progenitor cells, which can be used for cell-free therapy as a substitute for cells to treat diseases considering the lack of limitations in working with cells [32].Given the potential abilities of exosomes derived from MSCs in the process of tissue repair and immune system modulation in autoimmune diseases and cancers, researchers have   developed a special and new approach for using exosomes as a cell substitute in the treatment of diseases [33].In terms of activity, exosomes are highly influenced by the conditions in which they exist.For example, from which person they were taken, from which type of cell they were extracted and under what inflammatory or anti-inflammatory conditions they were stimulated.MSCs secrete a large amount of exosomes under starvation, serum-free environment, and inflammatory or non-inflammatory conditions, showing different effects in each of these circumstances.Based on studies, it has been determined that if MSCs are exposed to inflammatory conditions, they show a stronger anti-inflammatory function, and as a result, it is expected that exosomes extracted from the cells under the same conditions will show actions similar to the parent cell.As part of our research, we looked into the expression of immunomodulatory genes in exosomes derived from Poly (I:C) treated hWJ-MSCs.We showed that the stimulation of WJMSCs with 50 μg concentration of Poly (I:C) as an inflammatory stimulus at different time intervals (12 h, 24 h and 48 h) increases the expression of anti-inflammatory genes galectin-1, galectin-3, galectin-9 and HGF in the derived exosomes.
Reesink et al. investigated the expression of galectin-1 and galectin-3 genes in exosomes derived from Interleukin-1 beta (IL-1β), Tumor necrosis factor-α (TNF-α)-primed Bone marrow-derived mesenchymal stem cell (BMMSC) and showed that the expression of these genes decreases in inflammatory conditions [34].The source of stem cells and inflammatory factors in this study was different from ours, which could account for the difference of results with our study.Bone marrow mesenchymal stem cells constitutively express galectin-1, 3, and 8, leading to the suppression of T-cells and alleviation of inflammatory reactions such as those seen in graft versus host disease (GVHD) and autoimmune disorders [14].In terms of the expression of anti-inflammatory genes galectin-1 and galectin-3 in MSCs, the mentioned study is in line with our results because we showed that when MSCs are in inflammatory conditions, the expression of galectin-1and galectin-3 increases in exosomes.In our research, Poly (I:C) increased the expression of galectin-9 in MSCs-derived exosomes.MSCs were highly stimulated to produce galectin-9 when exposed to several proinflammatory stimuli, including Interferon gamma (IFN-γ), TNF-α, and the ligands of Toll-like receptors (TLRs) TLR2, TLR3, Poly(I:C), and TLR4, Lipopolysaccharides (LPS) [16].Thus, they found that galectin-9 contributes to the inducible immunomodulatory functions of MSCs.Moreover, a study was conducted on the immunomodulatory effect of Umbilical cord mesenchymal stem cell (UC-MSC), which showed that among galectin-1, galectin-3, galectin-1, TGF-β, HGF, PGE2, galectin-8, the strongest immunomodulatory effect on human peripheral blood mononuclear cells (PBMCs) population was related to galectin-3 gene.However, other genes, including galectin-1, galectin-9 and HGF as targets of our study did not show a significant effect [35].No research has been carried out to examine the immunomodulatory effects of HGF gene in MSCs-derived exosomes under inflammatory conditions, but studies on cell therapy have been performed with MSCs, in which the HGF gene has been transduced into these cells.MSCs expressing HGF gene suppress alloreactive responses in corneal transplants, suppress antigen-presenting cell maturation in draining lymphoid tissue, limit the responses of Th1 cells, and reduce the infiltration of inflammatory cells to the transplanted tissue [36].A study in line with ours, showed that the expression of TNF-α, IDO, IL-1β genes was increased, while the expression of IL-10 was decreased.The expression of the genes for galectin-1 and TGF-β remained unchanged [37].Their result regarding the galectin-1 gene was contrary to our findings.To explain these differences, there are many reasons in relation to mesenchymal stem cells and exosomes, including diverse source of derived mesenchymal cells, various incubation times, different isolation methods of stem cells, difference of exosome extraction method, varying concentrations of the stimulating substance and variable experimental conditions.Stimulation of MSCs with TLR4 ligand (LPS) increased the expression of IL-10 and TGF-β genes in their exosomes, possibly suppressing immune responses [38].LPS-primed MSC-Exo enhances the immunosuppressive effects of exosomes through increasing the ratio of anti-inflammatory gene to pro-inflammatory cytokine, leading to the polarization of M1 macrophages to M2 anti-inflammatory phenotype [39].MSC-Exo increased IL-10 and TGF-β production by regulatory T-cells and subsequently stimulated the immunoregulatory function of these cells [40].Pierce et al. demonstrated that the immunomodulatory and antimicrobial proteomic profile of bone marrow mesenchymal stem cell-derived exosomes enhanced after priming with Poly(I:C) [41].TNF-α-primed MSC-Exo promoted macrophage polarization to M2 phenotype.Subsequently, they indicated that galectin-1 expression was upregulated in TNF-αprimed MSC-Exo, which could play the role of an immunosuppressor agent [42].There is certain limitation that should be acknowledged.
Our research was conducted to measure only the expression of galectins and HGF genes.We did not check the level of galectins and HGF proteins in exosomes derived from Poly (I:C) Treated WJ-MSCS and we did not explore the effects of these exosomes on myeloid or lymphoid cells.This limitation underscores the need for more comprehensive studies in the future.

Conclusion
Stimulation of TLR-3 in Wharton's Jelly-derived MSCs may enhance the anti-inflammatory properties of exosomes isolated from WJ-MSCs by increasing the expression of immune regulatory genes galectin-1, galectin-3, galectin-9, and HGF in exosomes.

Data avalibility statement
Data will be made available on request by email to me:amariafshin@yahoo.com.

Fig. 1 .
Fig. 1. Isolation and characterization of hWJ-MSC.(A) Morphology of WJ-MSCs after about four weeks (B) After 21 days, osteogenic differentiation was assessed by Alizarin Red S staining, calcium deposition was stained bright orange-red.(C) Oil Red O staining of WJ-MSCs, intracellular lipid accumulation was stained bright red in adipocytes at day 21.Original magnifications = 40 × , bar = 200 μm (A-C).

Fig. 2 .
Fig. 2. Flow cytometry analysis of surface markers of hWJ-MSC.Flow cytometry analysis showing that WJ-MSC were negative for CD34, CD31 and CD45 but positive for CD73, CD90 and CD105.Positive expression was based on isotype control (blue curve).

Fig. 3 .
Fig. 3. Characterization of exosomes isolated from WJ-MSCs.(A) Size, PDI and zeta potential of exosomes.Data presented as mean ± SD. (B) Dynamic light scattering (DLS) indicated that the mean size of isolated exosomes was 132 nm.

Fig. 4 .
Fig. 4. Characterization of exosomes by SEM.Scanning electron microscopy (SEM) images indicated that exosomes had a uniform spherical shape with no significant deformities.

Fig. 5 .
Fig. 5. Flow cytometry analysis of surface markers of exosomes.Flow cytometry analysis showing that exosomes were negative for CD9, CD63 and CD81.Positive expression was based on control (red curve).

Fig. 6 .
Fig. 6.Toxicity of Poly (I:C) on WJ-MSCs.The viability was greater than 86 % after 12, 24, 48, at different concentration of Poly (I:C).Mean ± SD was used to represent the data.